BritishJournal ofOphthalmology 1994; 78: 386-389

Toxic side effects of local anaesthetics on the humancornea

M Boljka, G Kolar, J Vidensek

Department ofOphthalmology,University Ljubljana,SlovenijaM BoljkaG KolarJ VidensekCorrespondence to:Dr M Boijka, Okulisticnaklinikia, Ljubljana 61000,Zaloska 2, Slovenija.Accepted for publication13 December 1993

AbstractThe cytotoxic effects of 05% amethocaine(tetracaine) on the human cornea were investi-gated by scanning electron microscopy. Theultrastructural examination of epithelial cellsshowed damage of the cell membrane, rare-

faction and loss of microvilli, deposits ofamethocaine on the corneal surface and accel-erated desquamation of superficial epithelialcells.(BrJ Ophthalmol 1994; 78: 386-389)

Figure The corneal surface following application of0 5% amethocaine. Numerousamethocaine deposits ofvarious sizes (0-2-3-0 rim) are seen. A deposit being engulfed (arrow)(SEM x2200).

Figure 2 Higher magniJication of tigure 1. Deposit (arrow) engulfed into the damaged cellmembrane ofa 'dark' cell devoid or microvilli (SEM x 10 000).

Local anaesthetic drugs have the ability revers-ibly to block conduction of nerve impulses at theaxonal membrane level. They diffuse throughepithelial cells to reach the corneal nerves via thenodes of Ranvier. Because of their direct interac-tion with the specific binding sites within the Na+channels in the axonal membrane, local anaes-thetics interfere with the Na+ influx through themembrane and reduce electrical excitability.

HistoryThe first local anaesthetic to be discovered wascocaine, an alkaloid contained in the leaves ofErythroxylon coca, first isolated by Niemann in1860.2 The clinical use of cocaine in ophthal-mology was initiated by Koller3 and Knapp' in1884. Soon after its surface anaesthetic propertyhad been recognised, cocaine applied to thecornea was found to cause epithelial erosion.5 Forthat reason a chemical search for syntheticcocaine substitutes started. One of syntheticcocaine substitutes is 0 5% amethocaine, whichis five to eight times stronger than cocaine,6 itslocal anaesthesia is produced within 25 secondsand lasts for up to 15 minutes.

All lpcal anaesthetics have severe toxic sideeffects after long term, uncontrolled overuse.Their toxicity was reported in patients withanaesthetic induced keratopathy.''2 Commonfindings were extensive erosions and persistentepithelial defects. Perforating keratoplasty waseven required to treat corneal perforation orsevere corneal opacities,'3 14 and enucleation hadto be performed to relieve intractable pain. 5Only animal (rabbit,'6 rat'7) eyes are suitable

for the investigations of pharmacodynamicproperties of local anaesthetics, as they allowsuccessive assessments of biochemical andmorphological alterations of living cells. Noreport has been found on scanning electronmicroscopic investigation of the human corneaexposed to local anaesthetics, so in our study weused a human cornea.

Materials and methodsThe side effects of 0 5% amethocaine werestudied on two normal human corneas obtainedby enucleation of eyes because of malignantchoroidal melanoma. During the operation, thecorneas were moistened constantly with a solu-tion of 0 9% NaCl. Immediately after enuclea-tion, the corneas were excised and halved. Thefirst half was instilled with one drop of 05%amethocaine three times at 2 minute intervals,while the second halfofthe same cornea served asa control and was instilled with 09% NaCl only.After 6 minutes, both halves of the same corneawere put into 2% glutaraldehyde for 2 hours and

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Figure 3 Large deposits on a 'dark' cell devoid ofmicrovilli. Tiny and numerous deposits onmicrovilli ofthe adjacent cell (SEM x 7500).

Figure 4 A control SEM: the other halfofthe same cornea instilled only with 0 9% NaCl.A characteristic pattern ofnornal superficial cells. A 'dark' cell (arrow) (SEM x 1400).

Figure 5 Higher magnification ofa detail ofFigure 4, explaining the phenomenon ofthe'dark' cell. The dark-light contrast ofthe cell is still visible (SEM x3700).

prepared for scanning electron microscopy(SEM). Another human cornea was similarlyprepared. We examined a further two humancorneas with SEM, so we had four controlspecimens.

ResultsThe cytotoxicity of0 5% amethocaine was evalu-ated according to morphological changes of theepithelial corneal cells.

AMETHOCAINE DEPOSITSAmethocaine deposits were present on microvilli(Fig 1) or on cell membrane devoid of microvilli(Figs 2 and 3). The deposits were of differentsizes, round or lump-shaped, and scattereddensely over numerous cells ofthe corneas. Theyappeared only after instillation of 0 5% ametho-caine as they were not seen on the control corneasinstilled with just 09% NaCl (Figs 4, 5, and 6).

LOSS OF MICROVILLISome cells showed rarefaction of microvilli whilenumerous cells were completely devoid of them(Figs 2 and 3). A comparison with undamaged,control cells (Figs 4, 5, and 6) suggested that theloss of microvilli was the result of the installationof 05% amethocaine.

INCREASED DESQUAMATIONThe major cytotoxic effect of amethocaine wasincreased desquamation of 'light' cells (Fig 7).SEM demonstrated some superficial cells at themoment of their desquamation (Fig 8).

DAMAGE TO THE CELL MEMBRANEAt higher magnifications, ruptures were seen onthe desquamated cells (Fig 8).

CONTROLThe damaged cells were compared with thenormal ones. Figures 4, 5, and 6 showed normalsuperficial cells of the human cornea. It waspossible to make an ideal comparison betweentwo halves of the same cornea: one half instilledwith 05% amethocaine and another instilledwith 09% NaCl. The small number of 'dark'cells (Fig 4) indicates an intact corneal surfacewith a normal desquamation rate.The phenomenon of the 'dark' cell, explain-

able by the difference between microvilli, ispresent in Figures 4, 5, and 6. Microvilli of the'dark' cell appear flattened, as they were com-pressed by the overlying older 'light' cell beforedesquamator of this 'light' cell occurred.

DiscussionThe main effect of ocular local anaesthetics is theprevention ofpain, but their toxic side effects areresponsible for damage to epithelial cells. Whilethe process involved in pain blocking is wellunderstood, the molecular and cellular mecha-

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Boljka, Kolar, Vidensek

Figure 6 Higher magnification ofa detailfrom Figure5. The 'darkness' ofthe cell (arrow)is hardly visible. Normal microvilli (SEM x 12 000).

Figure 7 Corneal surface following application of0 5% amethocaine. An increaseddesquamation of 'light' cells. A desquamated cell in the middle (SEM x300).

Figure 8 Higher magnification ofFigure 7. The desquamated epithelial cell damaged by0-5% amethocaine, shows a folded surface and disrupted membrane (SEM x 1600).

nisms ofthe toxicity oflocal anaesthetics have notyet been satisfactorily clarified. 8

In our study the toxic effects of amethocainewere seen on the cell membrane and microvilli ofepithelial cells. Amethocaine deposits demon-strated by SEM in Figures 1, 2, 3, and 8represented a morphological substrate of theinteraction between amethocaine and proteins ofthe cell coat. The engulfing of deposits into thecytoplasm observed in some cells (Fig 2) may beone of the ways in which the amethocainepenetrates the cytoplasm, thus causing damageof the cell membrane and, finally, cell death(Fig 8).

Rarefaction and loss of microvilli may be alsothe consequence of toxic effects of amethocainedeposits on the microvilli (Figs 1, 2, and 3). Thedamage of microvilli leads to the increaseddesquamation, demonstrated by SEM as adecrease in the number of 'light' cells (Fig 7). It isknown that, during the process ofdesquamation,the interdigitation ofmicrovilli is diminished andmany contacts between them are interrupted.'719Similar alterations to microvilli were demon-strated by SEM in the rabbit cornea followingexposure to ultraviolet radiation.20 One of thereasons for the toxicity of amethocaine is changein pH. The most commonly used local anaesthe-tics belong to the benzoic acid ester group, andare weak bases - for example, 0 5% amethocainehas a pH of 3-7 to 6-0.

Reversibility of toxic effects of local anaesthe-tics can be demonstrated only in animals. Thus,toxic effects of one drop of oxybuprocaine(Novesin) instilled in a rabbit cornea are revers-ible within 60 minutes after instillation. 16 Leuen-berger,'7 who used SEM to study the toxicity oflocal anaesthetics applied to the rat cornea five to15 times at intervals of 30 minutes, reported adecreased number of 'light' cells and loss ofmicrovilli. His transmission electron micro-scopic (TEM) studies revealed a reduced numberof desmosomes, and ruptures of cell membraneand vacuoles in the cytoplasm. Harnisch16reportedTEM studies on side effects ofanaesthe-tics on the epithelial cells of the rabbit cornea,where he revealed all stages of cell degeneration,including rarefaction of microvilli.

Toxicity of local anaesthetics depends on theirrate of elimination. Despite the very slow rate ofhydrolysis of local anaesthetics, their toxicity issoon diminished by a continuous flow of tears,washing away amethocaine and cells contami-nated with the anaesthetic. Local anaesthetics areknown to delay healing of epithelial cornealdefects because they inhibit epithelial sliding.2'The comparison between SEM and slit-lamp

observations of anaesthetised cornea is interest-ing; both show the same cytotoxic effects of localanaesthetics on the patient's cornea. By slit-lampvery small, white dots are visible, representinggroups of denaturated superficial cells; the patho-logical prolongation of breakup time (BUT) is aresult of the increased cell desquamation.Adverse side effects of a single instillation of a

local anaesthetic are always mild and reversible in1-3 hours. However, anaesthetic inducedkeratopathies are extremely rare, despite exten-sive use oflocal anaesthetics in everyday ophthal-mic practice.

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The authors thank-Professor Dr N Pipan-and Dr M Psenicnik fortheir help in scanning electron microscopic examinations.

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